Discovery of an antibacterial isoindolinone-containing tetracyclic

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Discovery of an antibacterial isoindolinone-containing tetracyclic polyketide by cryptic gene activation and characterization of its biosynthetic gene cluster Wei Li Thong, Kazuo Shin-ya, Makoto Nishiyama, and Tomohisa Kuzuyama ACS Chem. Biol., Just Accepted Manuscript • DOI: 10.1021/acschembio.8b00553 • Publication Date (Web): 06 Aug 2018 Downloaded from http://pubs.acs.org on August 7, 2018

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ACS Chemical Biology

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Discovery of an antibacterial isoindolinone-containing tetracyclic polyketide by cryptic

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gene activation and characterization of its biosynthetic gene cluster

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Wei Li Thong,1,4 Kazuo Shin-ya,3 Makoto Nishiyama,1,2 and Tomohisa Kuzuyama*1,2

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Biotechnology Research Center and 2Collaborative Research Institute for Innovative Microbiology, The

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University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan

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135-0064, Japan

National Institute of Advanced Industrial Science and Technology (AIST), 2-4-7 Aomi, Koto-ku, Tokyo

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(MIB), The University of Manchester, 131 Princess Street, Manchester M1 7DN

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*Corresponding author: [email protected]

Present address: School of Chemistry & Manchester Institute of Biotechnology John Garside Building

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ACS Paragon Plus Environment

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Abstract

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The major setback in natural product screening is the decreasing hit rate of novel bioactive compounds

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containing new chemical skeletons. Here we report the identification and biosynthesis of isoindolinomycin

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(Idm), an unprecedented bioactive polyketide with a novel isoindolinone-containing tetracyclic skeleton.

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Idm was discovered through the screening of rifampicin-resistant (rif) mutants that were generated from 9

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actinomycete strains used in this study. Of the 114 rif mutants isolated, the mutant S55-50-5 was found to

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overproduce Idm, which is almost undetectable in the wild type Streptomyces sp. SoC090715LN-16. An in

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silico analysis coupled with gene deletion experiments revealed a biosynthetic idmB gene cluster that is

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responsible for the production of Idm. The biosynthetic studies of Idm primarily focused on the formation

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of the 5-membered ring in the tetracyclic structure and the attachment of the methyl group to the core

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structure. In addition, the malachite green phosphate assay performed using a stand-alone adenylation

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domain (idmB21) demonstrated involvement of glycine in the formation of the isoindolinone-containing

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skeleton. This study contributes to an increase in the structural diversity of polyketides and paves the way

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toward an understanding of the complete biosynthetic pathway of a novel class of tetracyclic polyketides.

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INTRODUCTION

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For the past 50 years, antibiotic research has provided us with thousands of microbe-derived bioactive

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compounds with a variety of chemical structures, such as polyketides, peptides and terpenes. Many of these

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bioactive compounds are isolated from actinomycetes.1 In fact, actinomycetes are still an important source

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for the production of clinically used naturally derived drugs.2 Nevertheless, the discovery of new bioactive

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compounds from actinomycetes has been decreasing since the late 1970s.3 Moreover, despite the urgent need

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for antibiotics to fight “superbugs”, many major pharmaceutical companies are abandoning antibiotic

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research.4 One of the reasons is that antibiotic development is economically burdensome because finding a

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lead compound that can act as an antibacterial agent is difficult.5

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The genomic era has yielded many published genome sequences, which revealed that an

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actinomycete strain contains more than 20 biosynthetic gene clusters predicted to encode different

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structural classes of secondary metabolites.6-8 Only a small number of these biosynthetic gene clusters are,

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however, expressed under standard culture conditions, and in many cases, they are involved in the

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production of known compounds. The remaining gene clusters are classified as cryptic or orphan because

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the metabolites produced by these gene clusters are unidentified. To address this problem, considerable

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research has been dedicated to activating the cryptic gene clusters during culture.9 To identify new bioactive

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compounds, we used a cryptic gene activation strategy based on screening for rifampicin-resistant (rif)

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mutants.10 This method previously proved effective when we identified 2 novel methylbenzene-containing

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polyenes.11 Using the same method, we continued to screen for rif mutants from 9 actinomycete strains with

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known draft genome sequences. Here, we describe the screening of the rif mutants that led to the

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identification of a novel isoindolinone-containing tetracyclic polyketide, named isoindolinomycin and its

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biosynthetic pathway based on bioinformatics analysis coupled with gene deletion experiments.

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RESULTS AND DISCUSSION

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Isolation of rifampicin-resistant (rif) mutants and comparative metabolic screening. Nine

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actinomycete strains with known draft genome sequences were used in this study (Table S1). The

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generation of rif mutants was achieved at 5-fold, 10-fold, and 50-fold excesses of the minimum inhibitory

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concentration (MIC) of rifampicin for each actinomycete strain. The MIC and rifampicin concentrations

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used to generate mutants are summarized in Table S2. Rifampicin is a potent broad-spectrum antibiotic that

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inhibits bacterial DNA-dependent RNA synthesis.12 Consistent with previous reports, rifampicin was found

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to be potent against most of the actinomycete strains when used at or below 3.125 µg mL–1.

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The generation of spontaneous rifampicin resistance in the 9 actinomycete strains resulted in the

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isolation of a total of 114 rif mutants (Table S3). Of these rif mutants, only 36 mutants that developed at the

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50-fold MIC level were selected for the following comparative metabolic screening process. This is based on

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our previous work, where rif mutants that developed at high concentrations of rifampicin exhibited greater

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overproduction than those that developed at lower concentrations.11 Additionally, Ochi et al. reported that

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mutants resistant to high drug concentrations are more stable in their phenotype.13 The selected rif mutants

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were screened for overproduced compounds by comparing their metabolic profiles with that of the parent

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strain. Of them, a 50-fold MIC rif mutant (S55-50-5), derived from Streptomyces sp. SoC090715LN-16

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(S55), produced some metabolites that were almost undetectable in the parent strain when cultivated in

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different media (Figure 1A). Isolation and structural elucidation of these metabolites were performed, but

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only the structure of metabolite 1 was successfully elucidated due to the low concentrations of the remaining

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metabolites. Metabolite 1 was named isoindolinomycin (Idm) after confirming it as an unprecedented

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bioactive compound (Figure 1B).

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Additionally, mutation analysis of the rpoB gene of S55-50-5 identified a point mutation that

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occurred in position C1309T (Figure S1A) that replaced the histidine residue at position 437 with tyrosine 4


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(Figure S1B). Tanaka et al. list rpoB mutations that are effective for antibiotic overproduction.14 The report

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emphasizes that mutations at amino acid residue 437, notably H437Y, are often effective in a wide variety of

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actinomycetes.

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Structural elucidation. Idm was isolated as a reddish orange amorphous solid with the molecular

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formula C24H26NO8Cl, as determined by HRESI-MS (m/z 492.1417 [M+H]+ calculated for C24H27NO8Cl+:

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492.1420). The presence of the M+2 signal in the mass spectrum for Idm indicated the presence of a

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chlorine atom (Figure S2). The structure of Idm was then elucidated by extensive NMR spectral analyses

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(Figure S3–S14). The 1H NMR spectrum (600 MHz, chloroform-d) of Idm showed 17 signals that were

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assigned to 3 aliphatic methylene units, 5 methine signals (δ 2.67, 2.89, 3.72, 4.22 and 4.89), an aromatic

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proton (δ 7.63), an exchangeable NH (δ 6.79), a hydrogen-bonded phenolic OH group at δ 14.37, two

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aliphatic methyl signals (δ 0.98 and 1.41), an anomeric proton (δ 4.74), and a methoxy signal (δ 3.40)

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(Table 1). The

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(C-1 and C-9), an aromatic methine (C-5), and 10 aromatic quaternary carbons, 2 of which (C-10 and C-11)

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could be assigned to aromatic carbons attached to oxygen atoms. In the aliphatic region, 3 methylenes (C-2,

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C-7 and C2’), 5 methine carbons (C-3, C-3’ C-4, C-4’, and C-5’), 2 methyl carbons (C-12 and C-6’), an

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anomeric carbon (C-1’), and a methoxy carbon (C-7’) were detected.

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C NMR and HSQC spectra indicated the presence of 24 carbons, including 2 carbonyls

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The structure of Idm was then determined by 2D NMR experiments (COSY, HSQC, HMBC and

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NOESY). Analysis of the 1H-1H COSY spectrum showed the correlations of the proton sequence from H-1’

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to H-6’ to be indicative of a deoxy sugar moiety (Figure S15). The HMBC correlations from H-4’ to the

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methoxy carbon C-7’ then determined the deoxy sugar moiety to be 4-O-methyl-2,6-dideoxyhexopyranose.

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Another fragment of the H-2/H-3/H-4 and H-3/H-12 correlations was also observed in the 1H-1H

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COSY spectrum. The HMBC correlations of this partial structure to a carbonyl group (H-2 to C-1), an

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aromatic quaternary carbon (H-2 and H-4 to C-11a) and an aromatic methine (H-4 to C-5) indicated that it 5



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was attached to an aromatic ring. The presence of the aromatic ring was confirmed by the HMBC

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correlations observed from H-5 to C-11a and C-10a. HMBC correlations were also observed from the

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highly de-shielded 11-OH to C-10a, C-11 and C-11a, indicating that it is a phenolic ring. However, the

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HMBC connectivity observed in Idm ended at the correlation from H-5 to C-6 (Figure S15). Furthermore,

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no correlation to H-7 was observed, leaving the structure only partially elucidated.

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To solve this problem, NMR analyses for Idm in different solvents were performed. As a result,

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the missing correlations from the H-7 signal were detected in NMR analyses performed using acetone-d6

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(Table 1). The HMBC correlations from H-7 to C-6, C-6a, C-9 and C-9a established the presence of a

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5-membered ring fused with the other 2 aromatic rings (Figure S15). Furthermore, the relatively

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downfield-shifted signals of the methylene C-7 in both 1H and 13C-NMR indicated that it is attached to an

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amine moiety within the ring system. The NOESY correlations were interpreted using the spectrum

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recorded in chloroform-d, due to overlapping of the acetone-d6 solvent peak in the aliphatic region. Thus, a

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planar structure of the aglycone of Idm was established and the relative stereochemistry of the aglycone

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was further determined from the 3JH2,H3 and 3JH3,H4 coupling constants and NOE correlations among 2-Ha,

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3-CH3, and 4-H (Figure 1B, Figure S16). The lack of NOE correlation between 2-Hb and 4-H supports this

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relative stereochemistry assignment. The 3JH1’,H2’b coupling constant (9 Hz) indicates that a linkage between

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the aglycone C4 and the deoxy sugar C1’ is a β-glycosidic bond. The NOE correlation observed between

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1’-H and 4-H is consistent with the configuration of the C1’ anomeric carbon.

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To the best of our knowledge, Idm represents the first isolated natural product having a

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tetracyclic structure with a unique isoindolinone skeleton. Structure searches performed using SciFinder

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(American Chemical Society) returned results indicating that this isoindolinone-containing tetracycle is a

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novel natural product skeleton. Moreover, the linear 6-6-6-5 ring system coupled with the non-aromatic

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D-ring of Idm is a rare feature for polyketides assembled by type II polyketide synthase (PKS) systems.

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The compound with the most similar structure to the Idm core structure is lactonamycin, which was

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isolated from Streptomyces rishiriensis MJ773-88K4 (Figure 1B).15

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Biological activity. The isolated Idm was tested for cytotoxicity against the SKOV-3, Meso-1,

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and Jurkat cell lines and for antimicrobial activity. Idm was cytotoxic to all the tested cell lines, the IC50

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values in SKOV-3, Meso-1 and Jurkat cells were 19.2 µM, 7.35 µM and 8.33 µM, respectively (Figure

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S17A). Idm also exhibited inhibitory activity against Staphylococcus aureus at an IC50 of 11.6 µM (Figure

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S17B), but showed no inhibitory activity against Escherichia coli, Bacillus subtilis or Micrococcus luteus

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(Figure S17C). Tetracycles such as anthracycline and tetracycline are well-known classes of antibiotics.

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Despite the potency of these drugs, which bind specifically to bacterial ribosomal subunits, they are

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currently facing efflux pump type resistance as a consequence of their overuse.16 Idm has a unique

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tetracyclic skeleton that may offer new binding possibilities in the active sites of the drug targets.

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Identification of the isoindolinomycin biosynthetic gene cluster. A point of interest in the

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biosynthesis of Idm is the formation of the unique isoindolinone-containing tetracyclic structure. Based on

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the structure of Idm, an amino acid, presumably glycine, is predicted to act as the starter unit in the type II

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PKS system. Precursor incorporation experiments of lactonamycin, the known compound most similar to

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Idm, show that its biosynthesis involves glycine or its derivative.17 However, no further evidence was

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provided for the enzyme or the actual substrate to support this observation. Therefore, to investigate this

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6-6-6-5 tetracyclic ring system, an in silico analysis of the draft genome sequence of Streptomyces sp.

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SoC090715LN-16 was performed. Use of a flavin-dependent halogenase homolog (Protein ID: AFV71318)

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as a search query returned two biosynthetic gene clusters, each bearing a gene with close homology to

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tryptophan halogenase (Figure S18). Analysis of the flanking regions in these gene clusters revealed that

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the idmB gene cluster responsible for Idm production contains genes encoding type II PKS (idmB17, 18),

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glycosyltransferase (idmB30) and methyltransferase (idmB4, 24, 39), the presence of which are highly

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rational for the biosynthesis of Idm (Table 2, Figure 2). To confirm this assumption, we disrupted idmB32

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in the gene cluster (Figure S19) because idmB32 shows close homology to tcmG, a dioxygenase

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indispensable for the biosynthesis of tetracenomycin (Tcm) that catalyzes the triple hydroxylation of the

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tetracyclic TcmA2 to TcmC.18 As expected, the deletion abolished the production of Idm, verifying that the

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gene cluster is responsible for Idm biosynthesis (Figure S19). Analysis of the genes in the idmB

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biosynthetic gene cluster revealed that many of them are homologous to those in the biosynthetic gene

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cluster of lactonamycin (Table 2). We speculate that the identified homologous genes primarily encode

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proteins that participate in the formation of the polyketide skeleton, such as type II PKS, polyketide cyclase,

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and some of the tailoring enzymes. This speculation is based on the observation that Idm and lactonamycin

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share some similarity in their core skeletons.

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Loading of the starter unit. With the identified biosynthetic gene cluster in hand, we proceeded

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to investigate the isoindolinone-containing core skeleton. We speculated that the starter unit glycine must

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be adenylated prior to loading onto an acyl carrier protein (ACP) and is subsequently incorporated into the

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polyketide chain backbone (Figure S20). A recent report on streptothricin biosynthesis revealed 2

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stand-alone adenylation (A) domains that each catalyze a different type of reaction: the adenylation of the

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starter unit L-β-lysine for PCP loading and the adenylation of L-β-lysine for the elongation of the

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L-β-lysine peptide chain. Therefore, the adenylation reaction in Idm biosynthesis is most likely catalyzed

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by the stand-alone adenylation domain (idmB21) found in the gene cluster. Sequence analysis of idmB21

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revealed that the signature code for this gene product is “DLFFLCLLSK” and the web software

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NRPSpredictor2 predicts the recognition of glycine or alanine residues.20 To determine the substrate for

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IdmB21, the malachite green phosphate assay was performed. The recombinant IdmB21 protein was

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purified (Figure S20) and used in the in vitro assay with 20 amino acids. The reaction products were then

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tested with the malachite green phosphate kit to measure phosphate liberated from the degradation of ATP

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during the adenylation reaction. IdmB21 displayed a strict specificity for glycine (Figure S20), confirming

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the involvement of glycine as the likely starter unit for the polyketide chain backbone of Idm.

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Methylation in isoindolinomycin biosynthesis. We were intrigued by the methylation of the

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non-aromatic D-ring of Idm. To investigate the methylation mechanism, we deleted the three putative

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methyltransferase genes (idmB4, idmB24, and idmB39) found in the idmB gene cluster (Figure 2, Figure

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S21). The deletion of idmB4 abolished the production of both Idm and its aglycone (Figure 3A), indicating

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that IdmB4 may be the enzyme catalyzing the transfer of the methyl substituent to the core structure. Using

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this method, we expected the accumulation of an intermediate that allowed for the elucidation of the

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reaction pathway, but no intermediate was detected. This result may be due to several factors, including the

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potential instability of the intermediates, and that methylation by IdmB4 occurs during the generation of the

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polyketide chain. If the latter reason is proven true, the reaction catalyzed by IdmB4 would represent an

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unprecedented methylation mechanism in aromatic polyketide biosynthesis.

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The deletion of idmB39 revealed that it may be responsible for the O-methylation of the sugar

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moiety. This deduction is based on the observation that Idm production was abolished and an unknown

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metabolite, most likely an Idm lacking a methyl group in its deoxy sugar moiety, was detected (Figure 3B).

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Moreover, the detection of the Idm aglycone in the deletion mutant further confirmed that idmB39 is not

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responsible for the transfer of a methyl moiety to the tetracyclic structure (Figure S22). The Idm aglycone

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was detected in the culture extract of S55-50-5 but due to its low yield, the structure could only be

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predicted from the LC-MS/MS spectra (Figure S23).

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Unlike idmB4 and idmB39, the deletion of idmB24 did not terminate the production of Idm

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(Figure S24A). Although this result suggests that idmB24 may not be related to the biosynthesis of Idm, an

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unknown metabolite with the UV spectrum that is indicative of an Idm-related compound and putative

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molecular formula C19H13NO6 was accumulated in the deletion mutant (Figure S24B and C). The isolation

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and structural elucidation of this metabolite may provide information on the function of this enzyme.

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Proposed biosynthetic pathway. In silico analysis combined with gene deletion experiments led

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to the identification of the idmB gene cluster. Similar to all other type II PKS systems, idmB gene cluster

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has a set of minimal PKS with β-ketoacyl synthases, IdmB17 (KSβ) and IdmB18 (KSα), and IdmB25

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(ACP). IdmB18 has the acyltransferase GHSxG motif and a conserved Cys169 for acyl binding that are

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both characteristic of KSα, whereas IdmB17 has Gln characteristic of KSβ instead of the Cys.21 The

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biosynthesis of the isoindolinone-containing core structure is proposed to start with the adenylation of

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glycine by IdmB21 before the condensation and extension of 7 acetate units by IdmB17 and IdmB18. After

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the polyketide chain is assembled, cyclization reactions occur, presumably mediated by the polyketide

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cyclases IdmB19, IdmB20 and IdmB22 (Figure 4). Among these cyclases, IdmB22 exhibits high similarity

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to the N-terminal of the didomain cyclase TcmN, also known as an aromatase that catalyzes the cyclization

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reaction and subsequent elimination of water to produce the first aromatic ring.22 Thus, we suspect that

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IdmB22 functions as ketoreductase that is absent in the idmB gene cluster and that the remaining cyclases

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are responsible for the introduction of the remaining rings. The idmB gene cluster contains two ACPs,

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IdmB23 and IdmB25. IdmB25 exhibits higher identity (37%) to TcmM, an ACP from the minimal type II

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PKS in the biosynthesis of tetracenomycin.23 This suggests that IdmB25 may be part of isoindolinomycin

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minimal PKS. As IdmB23 shows only 21% identity to TcmM, we suspect that it is involved in the loading

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of glycine onto IdmB18. The presence of a thioesterase (IdmB12) is rarely associated with type II PKS

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systems. Such discrete thioesterases (TE), also known as type II TE, are typically found in modular type I

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PKS and non-ribosomal peptide synthetase (NRPS) systems.24 However, there are also cases reported

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where an acyltransferase homolog possessing thioesterase activity is involved in the type II PKS systems,

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producing non-acetate-primed polyketides. These acyltransferase homologs, such as ZhuC from the R1128

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PKS and EncL from the enterocin PKS, remove the usual acetate starter units that compete with the

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alternate alkyl acyl primer units.25,26 Therefore, it is conceivable that IdmB12 may have a similar function

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to type II thioesterases in preventing the initiation of Idm synthesis with acetate. Following the cyclization

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reactions, a series of tailoring reactions including halogenation by IdmB26, dioxygenation by IdmB32,

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quinone reduction by IdmB37, and methylation by IdmB4 are expected to occur to form the Idm aglycone.

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The biosynthesis of the deoxy sugar moiety was also predicted by bioinformatics analysis. IdmB8,

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9, 28, and 29 are likely involved in the formation of dTDP-2,6-dideoxyhexopyranose from α-D-glucose

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1-phosphate. The idmB gene cluster, however, lacks a dTDP-glucose 4,6-dehydratase homolog.

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dTDP-glucose 4,6-dehydratase is a common enzyme involved in the irreversible deoxygenation at C6 to

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produce the key intermediate, 4-keto-6-deoxy-D-hexose for the biosynthesis of all 6-deoxysugars.27

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Although the idmB gene cluster does not possess 4,6-dehydratase, a putative dTDP-glucose 4,6-dehydratase

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was found in a different locus from the idm gene cluster on the S55-50-5 genome. Following the

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biosynthesis of dTDP-2,6-dideoxyhexopyranose, a glycosidic bond between the deoxy sugar moiety and

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the Idm aglycone could be formed by the Idm30 glycosyltransferase. Finally, the IdmB39

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O-methyltransferase catalyzes 4-O-methylation of the deoxy sugar moiety to produce Idm.

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CONCLUSION

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The aim of this study is to explore the hidden potential of actinomycetes via the activation of cryptic

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gene clusters. The aim was achieved when Idm was isolated from the rif mutant S50-50-5. This

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unprecedented compound possesses a novel skeleton with potent bioactivity and its chemical structure has

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steered our research towards the discovery of possible new biosynthetic mechanisms. Although the full

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biosynthetic pathway of the novel compound has yet to be elucidated, this report demonstrates the potential

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of actinomycetes as a source for the discovery of natural products. Naturally, there is no single method that

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can unlock all cryptic pathways in actinomycetes. Advances in genome sequencing coupled with software

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tools for genome annotation have allowed us to predict and identify potential novel natural products.

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However, in many cases, in silico screening works only for well-established natural products and is unable to

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recognize new types of biosynthetic assemblies. Moreover, the complex regulation of secondary metabolite

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biosynthesis constantly reminds us that there is more to be studied before we can understand the full potential

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of actinomycetes. The discovery of new synthetically challenging bioactive substances is expected from

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these microorganisms, and with them, we can contribute to the development of better disease therapies.

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METHODS

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Strains and oligonucleotides used in this study are listed in Table S1 and S4 of the Supporting Information

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section, respectively. The generation of rif mutants was performed on GYM agar containing different

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concentrations of rifampicin according to the MIC of each strain (Table S3). Rif mutants were cultured in

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A1-, GYM-, K- and A-94964-producing media for metabolite analysis. For large scale fermentation and

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isolation of Idm, the S55-50-5 mutant was cultivated in A-94964-producing medium. The extracts and

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fractions were analyzed using a UHPLC-PDA system (JASCO X-LCTM Systems, Tokyo, Japan) with a

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CAPCELLPAK C18 column (2.0 × 50 mm, Shiseido, Tokyo, Japan). Idm was finally purified using a

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preparative HPLC system (Senshu Scientific, Tokyo, Japan) with a Senshu Pak PEGASIL ODS column (20

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× 250 mm, Senshu Scientific, Tokyo, Japan). A high-resolution Triple TOF 5600 MS (SCIEX, Tokyo,

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Japan) equipped with a UFLC Nexera system (Shimadzu, Kyoto, Japan) was used with a CAPCELL PAK

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C18 column (2.0 × 50 mm, Shiseido, Tokyo, Japan) for HRESI-MS (positive mode). 1H, 13C and 2D NMR

256

spectra were recorded on a JEOL ECA-600 spectrometer (B0 = 14.01 T). Genome sequences were

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annotated

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(http://nocardia.nih.go.jp/fp4/),28 and 2ndFind (http://biosyn.nih.go.jp/2ndfind/). The specificity of the 12

with

protein

BLAST

(http://blast.ncbi.nlm.nih.gov/Blast.cgi),


FramePlot

4.0

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stand-alone adenylation domain was predicted using NRPSpredictor2 software.20 Gene inactivation was

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accomplished using the in-frame deletion method. The adenylation reaction of IdmB21 was determined in

261

vitro using the malachite green phosphate method. Detailed materials and methods are provided in the

262

Supporting Information section.

13



Page 14 of 25

Table 1. 1H (600 MHz) and 13C (150 MHz) NMR spectral data for Idm in chloroform-d and acetone-d6

Chloroform-d Position

δC

1

204.3

2a

40.3

Acetone-d6 δH, mult (J in Hz)

δC 204.3

2.43, dd (2.4, 18)

40.8

3.28, dd (4.8, 18)

2b

δH, mult (J in Hz)

2.49, dd (5.4, 18) 3.17, dd (4.8, 18)

3

33.8

2.67, m

34.2

2.57, m

4

76.2

4.89, d (3.6)

76.7

4.91, d (5.4)

4a

138.6

5

116.6

5a

136.7

138.2

6

113.5

112.0

6a

143.0

143.7

7

48.6

138.5 7.63, s

115.3

4.93, m

48.4

6.79 brs

NH

7.68, s

4.96, s 8.31 brs

9

171.7

171.2

9a

117.5

118.2

10

152.3

152.6

10a

115.9

115.1

11

164.0

163.3

11a

109.4

109.3 14.37, s

11-OH 12

17.1

0.98, d (7.2)

16.7

1.03, d (7.2)

1’

94.0

4.74, brd (9)

95.1

4.86, dd (1.8, 9.6)

2’a

36.6

2.02, brd (13.8)

37.3

1.97, ddd (1.8, 3.6, 13.8)

1.69, ddd (3, 9, 13.8)

2’b

1.66, ddd (3, 9.6 13.8)

3’

64.0

4.22, dd (3, 3)

63.0

4.26, dd (3, 3)

4’

82.5

2.89, dd (3, 9)

82.8

2.87, dd (3, 9)

5’

68.2

3.72, dq (6, 9)

68.2

3.76, dq (6, 9)

6’

18.4

1.41, d (6)

17.9

1.31, d (6)

7’

57.4

3.40, s

56.0

3.32, s

1

14


Page 15 of 25 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


2

Table 2. The deduced functions of the idmB gene products

1

Ami no Proposed Function acid s 110 Hypothetical protein

Hypothetical protein [Streptomyces sp. NRRL S-813]

28/39

WP_030186183

2

474

Transporter

Drug resistance transporter, EmrB/QacA subfamily [Streptomyces iranensis]

48/61

CDR06316

3

359

Aminotransferase

Aminotransferase [Streptomyces lividans 1326]

45/61

EOY48903

4

338

Methyltransferase

Lct35 [Streptomyces rishiriensis]

59/71

ABX71118

5

260

Dehydrogenase

Dehydrogenase [Streptomyces fulvoviolaceus]

51/62

WP_030619294

6

243

Regulatory protein

Regulatory protein TetR [Streptomyces iranensis]

50/65

CDR06320

7

452

Oxidase

FAD linked oxidase domain-containing protein [Amycolatopsis decaplanina]

50/62

WP_007031970

8

468

dTDP-2,3-dehydratase

Sim20 [Streptomyces antibioticus]

49/62

AAL15606

9

357

dTDP-1-glucose synthase

Putative dTDP-1-glucose synthase [Streptomyces vietnamensis ]

55/70

ADO32770

10

278

Oxidoreductase

LLM class flavin-dependent oxidoreductase [Streptomyces sp. URHA0041]

48/58

WP_033174223

11

451

Na: H Exchanger

Sodium:proton exchanger [Streptomyces sp. NRRL WC-3773]

60/74

WP_031005911

12

253

Thioesterase


42/52

ABX71117

13

449

Pyruvate carboxylase

Pyruvate carboxylase subunit A [Streptomyces iranensis]

76/86

WP_044581358

14

153

Biotin carboxyl carrier protein

Acetyl-CoA carboxylase biotin carboxyl carrier protein subunit [Streptomyces iranensis]

47/61

WP_052701348

15

571

Acetyl-CoA carboxylase

Acetyl-CoA carboxyl transferase [Streptomyces glaucescens]

57/69

WP_052413654

16

106

Monooxygenase


50/64

ABX71116

17

408

Beta-ketoacyl synthase (KSβ) Lct32 [Streptomyces rishiriensis]

68/77

ABX71115

18

419

Beta-ketoacyl synthase (KSα) Lct31 [Streptomyces rishiriensis]

80/86

ABX71114

19

130

Polyketide cyclase


61/74

ABX71113

20

116

Polyketide cyclase


60/69

ABX71112

21

510

Amino acid adenylation domain-containing protein


60/69

ABX71111

22

156

Polyketide cyclase


74/81

ABX71110

23

83

Acyl carrier protein


45/65

ABX71109

24

348

Methyltransferase


60/72

ABX71108

25

84

Acyl carrier protein


52/71

ABX71107

26

409

Tryptophan halogenase

FAD-binding monooxygenase [Streptomyces iranensis]

70/83

WP_044569677

27

638

Regulatory protein


44/60

ABX71106

28

334

dTDP-2,3-ketoreductase

dTDP-4-keto-6-deoxyhexose 2,3-reductase

67/79

BAD08364

29

265

dTDP-4-ketoreductase

dTDP-4-dehydrorhamnose reductase [Streptomyces yanglinensis]

49/60

SEG80896

30

412

Glycosyl transferase

Glycosyl transferase [Amycolatopsis mediterranei]

40/54

WP_013227238

31

139

Hypothetical protein

Hypothetical protein [Propionibacterium sp. KPL1844]

55/70

WP_023034785

32

491

Dioxygenase


52/62

ABX71124

33

107

Monooxygenase


44/62

ABX71116

34

106

Monooxygenase


45/68

ABX71125

35

247

Abhydrolase

Carboxymethylenebutenolidase [Streptomyces sp. AcH 505]

70/80

WP_041988759

36

93

Hypothetical protein

Hypothetical protein [Streptomyces sp. NRRL F-5126]

62/74

WP_030904778

37

313

Quinone reductase


56/67

ABX71126

38

279

Oxidoreductase

LLM class flavin-dependent oxidoreductase [Streptomyces sp. URHA0041]

45/59

WP_033174223

39

348

Methyltransferase

Methyltransferase [Streptomyces griseus]

36/47

WP_030766712

IdmB

Blast hit protein [Origin]

15


Identity/ Similarity (%)

Protein ID


3

Figure legends

4

Figure 1. (A) Comparative HPLC analysis of the metabolites from the culture extracts of Streptomyces sp.

5

SoC090715LN-16 (S55) and its rif mutant S55-50-5 in different media. (B) The structures of Idm

6

elucidated in this study and the structurally similar, previously reported compound lactonamycin.

7 8

Figure 2. Organization of the Idm biosynthetic gene cluster. The gene sequence is deposited in the DNA

9

database in the DNA databank under accession number LC386909.

10 11

Figure 3. Comparative HRESI-MS analysis of the culture extracts from S55-50-5, idmB4, and idmB39

12

deletion mutants. (A) The absence of Idm (a) and its aglycone (b) in the ∆idmB4 mutant. (B) The absence

13

of Idm (a) and the accumulation of a possible Idm derivative lacking a methyl group in its sugar moiety (b)

14

in the ∆idmB39 mutant.

15

16

Figure 4. The proposed biosynthetic pathway for isoindolinomycin.

17

18

Conflicts of interest

19

The authors declare no conflicts of interest.

20

21

ASSOCIATED CONTENT

22

Supporting Information Available: This material is available free of charge via the internet at

23

http://pubs.acs.org. Methods and Supplementary Figures, Tables, and References.

24

16


Page 16 of 25

Page 17 of 25 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


25

Acknowledgments

26

This work was supported by JSPS KAKENHI (16H06453 to T.K.), JSPS A3 Foresight Program, and the

27

Japan Agency for Medical Research and Development (AMED) (to K.S. and T.K.). W.L.T was supported by

28

the Ministry of Education, Culture, Sports, Science and Technology (MEXT) Japan.

17



29

References

30

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of silent biosynthetic gene clusters. Nat. Rev. Microbiol. 13, 509-523.

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10. Hu, H., Zhang, Q., and Ochi, K. (2002) Activation of antibiotic biosynthesis by specified

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mutations in the rpoB gene (encoding the RNA polymerase beta subunit) of Streptomyces lividans.

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Bentley, S. D., Chater, K. F., Cerdeno-Tarraga, A. M., Challis, G. L., Thomson, N. R., James, K.

Ikeda, H., Ishikawa, J., Hanamoto, A., Shinose, M., Kikuchi, H., Shiba, T., Sakaki, Y., Hattori,

Ohnishi, Y., Ishikawa, J., Hara, H., Suzuki, H., Ikenoya, M., Ikeda, H., Yamashita, A., Hattori,

Rutledge, P. J., and Challis, G. L. (2015) Discovery of microbial natural products by activation

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11. Thong,

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Methylbenzene-containing polyketides from a Streptomyces that spontaneously acquired

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rifampicin resistance: structural elucidation and biosynthesis. J. Nat. Prod. 79, 857-864.

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12. Calvori, C., Frontali, L., Leoni, L., and Tecce, G. (1965) Effect of rifamycin on protein

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synthesis. Nature 207, 417-418.

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13. Ochi, K., Okamoto, S., Tozawa, Y., Inaoka, T., Hosaka, T., Xu, J., and Kurosawa, K. (2004)

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Ribosome engineering and secondary metabolite production. Adv. Appl. Microbiol 56, 155-184.

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14. Tanaka, Y., Kasahara, K., Hirose, Y., Murakami, K., Kugimiya, R., and Ochi, K. (2013)

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Activation and products of the cryptic secondary metabolite biosynthetic gene clusters by rifampin

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resistance (rpoB) mutations in actinomycetes. J. Bacteriol. 195, 2959-2970.

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15. Matsumoto, N., Tsuchida, T., Nakamura, H., Sawa, R., Takahashi, Y., Naganawa, H., Iinuma,

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H., Sawa, T., Takeuchi, T., and Shiro, M. (1999) Lactonamycin, a new antimicrobial antibiotic

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produced by Streptomyces rishiriensis MJ773-88K4. II. Structure determination. J. Antibiot. 52,

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17. Zhang, X., Alemany, L. B., Fiedler, H. P., Goodfellow, M., and Parry, R. J. (2008) Biosynthetic

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investigations of lactonamycin and lactonamycin z: cloning of the biosynthetic gene clusters and

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18. Rafanan, E. R., Hutchinson, C. R., and Shen, B. (2002) Triple hydroxylation of tetracenomycin

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streptothricin biosynthesis. Nat. Chem. Biol. 8, 791-797.

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20. Röttig, M., Medema, M. H., Blin, K., Weber, T., Rausch, C., and Kohlbacher, O. (2011)

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K.,

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Classification

M.,

Framework

19


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Kuzuyama,

chemical

T.

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biology

of


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267, 19278-19290.

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22. Ames, B. D., Korman, T. P., Zhang, W., Smith, P., Vu, T., Tang, Y., and Tsai, S. (2008) Crystal

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24. Kotowska, M., and Pawlik, K. (2014) Roles of type II thioesterases and their application for

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25. Tang, Y., Koppisch, A. T., and Khosla, C. (2004) The acyltransferase homologue from the

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27. Lombó, F., Olano, C., Salas, J. A., and Méndez, C. (2009) Chapter 11. Sugar biosynthesis and

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107

20


Page 20 of 25

Page 21 of 25

A

B NMMP

A1

A1 medium

NMMP medium

7

O

1

1

OH OH 11

10

O 9

2

S55 S55 2

3

4

5

17 S55-50-5 S55-50-5

A270 nm

A270 nm

S55-50-5 S55-50-5

6

S55 S55 2

3

Retention time [min]

GYM medium

4

5

7'

O

NH

6' 12 3 4' 5' O 2'

3'

4

6

5

7 1

Cl

O

1'

OH

Isoindolinomycin

6

O

A-94964 producing medium 17

GYM

A-94964 producing medium

O

O

OH

O

A270 nm

S55 S55

O

S55-50-5 S55-50-5

S55-50-5 S55-50-5

3

4 5 Retention time [min]

6

3


6

O OH

O

S55 S55 2

N

O O

OH 2

7


A270 nm

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Lactonamycin

Figure 1. (A) Comparative HPLC analysis of the metabolites from the culture extracts of Streptomyces sp. SoC090715LN-16 (S55) and its rif mutant S55-50-5 in different media. (B) The structures of Idm elucidated in this study and the structurally similar, previously reported compound lactonamycin.



Page 22 of 25

1 kb

2

3

4 5 6

7

8

9

11

13

15

17 18

21

24

26

27

28

Type II PKS

Stand-alone A domain

Methyltransferase

Polyketide cyclase

Halogenase

Regulation

Post-polyketide modifier

Sugar biosynthesis

Other genes

30

32

39

Figure 2. Organization of the Idm biosynthetic gene cluster. The gene sequence is deposited in the DNA database in the DNA databank under accession number LC386909.


Page 23 of 25

A

B

a

Isoindolinomycin

a

Isoindolinomycin

XIC m/z 492.1425 [M+H]+ XIC m/z 348.0639 [M+H]+

0

idmB4

2


b

O

OH OH

Isoindolinomycin

8

10

Intensity

S55-50-5

Intensity

Intensity

XIC m/z 492.1425 [M+H]+

S55-50-5

0 2

0

2 4 4 6 6 8 Retention time [min] Retention time [min] O

0

00

88

OH OH

10

O

10 10

O

Cl

OH

XIC m/z 478.1269 [M+H]+

Intensity

idmB4 idmB39 44 66 Retention Retentiontime time[min] [min]

O

HO

S55-50-5 S55-50-5

22

8 10

NH

NH

+ OH m/zCl492.1425 [M+H] XIC XIC m/z 348.0639 [M+H]+

5

S55-50-5

idmB4 idmB39

b

O

Intensity Intensity

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


0

S55-50-5 idmB39 2


8

10

Figure 3. Comparative HRESI-MS analysis of the culture extracts from S55-50-5, idmB4, and idmB39 deletion mutants. (A) The absence of Idm (a) and its aglycone (b) in the DidmB4 mutant. (B) The absence of Idm (a) and the accumulation of a possible Idm derivative lacking a methyl group in its sugar moiety (b) in the DidmB39 mutant.



Adenylation HO O NH 2

IdmB23

ATP

ACP ACP

AMP O O

IdmB21 PPi

SH

AMP

S O

NH 2

IdmB 17 I8

N 2H

Glycine

O

+ IdmB25

CoA S Idm15 O

7x

Acetyl-CoA

Page 24 of 25

ACP

CoA

O

O

IdmB25 ACP

S NH 2

O

O

ACP

CoA SH S

O

O

OH OH OH

IdmB19, 20, 22

D

C

B

O A NH

O

S

O

O

O

O

IdmB26

HO

OH

Maonyl-CoA O

OH OH

O

O

OH OH

OH OH OH

O

IdmB37 NH OH

O

IdmB32 NH

Cl

O

NH

Cl

Cl

IdmB4 O

OH OH

O

O

IdmB30

OH OH

NH OH

IdmB28

O O

O

HO

Cl

OTDP

IdmB29

O OH

O

Cl

O HO

O

4,6-dehydratase

OH

OH OTDP

Sugar biosynthesis

OTDP

dTDP-2,6-dideoxyhexopyranose HOH 2C HO HO

O OH OTDP

OH OH

IdmB9

HOH 2C HO HO

O NH

O

O

O

Cl

OH

Isoindolinomycin

O

HO

OTDP

IdmB8

O

IdmB39

NH O OH

O

O

O OH OPO3H 2

α-D-Glucose 1-phosphate

Figure 4. The proposed biosynthetic pathway for isoindolinomycin.


Page 25 of 25

Rif mutant screening

Biosynthetic gene cluster NMMP

A1 7

S55 2

3

4

5

HPLC analysis Retention time [min]

O 7

OH OH S55-50-5

A270 nm

A270 nm

S55-50-5

6

2

3

4

O

5

NH

In vitro analysis HO O

6


O

Cl

O

NH 2

OH

GYM

A-94964 producing medium

7

Isoindolinomycin

rif S55-50-5 mutant A270 nm

S55-50-5

wild S55 type 2

O

S55

7

A270 nm

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


3


6

3


ATP

PPi

AMP O O

NH 2

Antibacterial assay

S55 2

IdmB21

6


Discovery of an antibacterial isoindolinone-containing tetracyclic

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